Molecular docking and MD simulation studies of 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine derivatives as novel inhibitors targeted to CDK2/4/6.

PURPOSE: Nowadays, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have been approved for treating metastatic breast cancer and have achieved inspiring curative effects. But some discoveries have indicated that CDK 4/6 are not the requisite factors in some cell types because CDK2 partly compensates for the inhibition of CDK4/6. Thus, it is urgent to design CDK2/4/6 inhibitors for significantly enhancing their potency. This study aims to explore the mechanism of the binding of CDK2/4/6 kinases and their inhibitors to design novel CDK2/4/6 inhibitors for significantly enhancing their potency in different kinds of cancers. MATERIALS AND METHODS: A series of 72 disparately functionalized 4-substituted N-phenylpyrimidin-2-amine derivatives exhibiting potent inhibitor activities against CDK2, CDK4 and CDK6 were collected to apply to this research. The total set of these derivatives was divided into a training set (54 compounds) and a test set (18 compounds). The derivatives were constructed through the sketch molecule module in SYBYL 6.9 software. A Powell gradient algorithm and Tripos force field were used to calculate the minimal structural energy and the minimized structure was used as the initial conformation for molecular docking. By the means of 3D-QSAR models, partial least squares (PLS) analysis, molecular dynamics (MD) simulations and binding free energy calculations, we can find the relationship between structure and biological activity. RESULTS: In this study, we used molecular docking, 3D-QSAR and molecular dynamics simulation methods to comprehensively analyze the interaction and structure-activity relationships of 72 new CDK2/4/6 inhibitors. We used detailed statistical data to reasonably verify the constructed 3D-QSAR models for three receptors (q2 of CDK2 = 0.714, R2pred = 0.764, q2 = 0.815; R2pred of CDK4 = 0.681, q2 = 0.757; R2pred of CDK6 = 0.674). MD simulations and decomposition energy analysis validated the reasonability of the docking results and identified polar interactions as crucial factors that influence the different bioactivities of the studied inhibitors of CDK2/4/6 receptors, especially the electrostatic interactions of Lys33/35/43 and Asp145/158/163. The nonpolar interaction with Ile10/12/19 was also critical for the differing potencies of the CDK2/4/6 inhibitors. We concluded that the following probably enhanced the bioactivity against CDK2/4/6 kinases: (1) electronegative groups at the N1-position and electropositive and moderate-sized groups at ring E; (2) electrogroups featured at R2; (3) carbon atoms at the X-position or ring C replaced by a benzene ring; and (4) an electrogroup as R4. CONCLUSION: Previous studies, to our knowledge, only utilized a single approach of 3D-QSAR and did not integrate this method with other sophisticated techniques such as molecular dynamics simulations to discover new potential inhibitors of CDK2, CDK4, or CDK6. So we applied the intergenerational technology, such as 3D-QSAR technology, molecular docking simulation techniques, molecular dynamics simulations and MMPBSA19/MMGBSA20-binding free energy calculations to statistically explore the correlations between the structure with biological activities. The constructed 3D-QSAR models of the three receptors were reasonable and confirmed by the excellent statistical data. We hope the results obtained from this work will provide some useful references for the development of novel CDK2/4/6 inhibitors.

Synthesis of organic solvent-free nitrogen-doped carbon quantum dots as unique green fluorimetric probes for analysis of abrocitinib in human plasma.

Atopic dermatitis (AD) is a persistent, inflammatory skin condition that impacts approximately 15 to 20% of children and 1 to 3% of adults globally. Common skin manifestations include papules, papulovesicular, and brown or red patches with swelling, crusting, and flaking. Therefore, the drug abrocitinib (ABR) was approved by the US FDA as an oral treatment for atopic dermatitis. The present study outlines the development of innovative, thermostable, and pH-stable organic solvent-free nitrogen-doped carbon dots (N@CQDs) synthesized through a one-step method for evaluating ABR with a notable quantum yield of 33.84% to minimize the use of organic solvents. Their cost-effectiveness, eco-friendly characteristics, and outstanding photocatalytic properties have established them as a promising alternative to conventional luminescent techniques like fluorescent dyes and luminous derivatization technique. The reaction of ABR with N@CQDs led to a significant decrease in the luminescent response of the produced green and stable carbon quantum dots at 513 nm. The detection range was determined to be 1.0-150.0 ng mL-1, with a lower limit of quantitation (LOQ) equal to 0.52 ng mL-1 based on the linear graph. The green method effectively used for analysis of ABR in pharmaceutical tablets and pharmacokinetic study with high sensitivity.

QbD Enabled Development and Evaluation of Pazopanib Loaded Nanoliposomes for PDAC Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the highly fatal types of cancer with high mortality/incidence. Considering the crucial role of vascular endothelial growth factor (VEGF) in PDAC progression, its inhibition can be a viable strategy for the treatment. Pazopanib, a second-generation VEGF inhibitor, is approved for the treatment of various oncological conditions. However, due to associated limitations like low oral bioavailability (14-39%), high inter/intra-subject variability, stability issues, etc., high doses (800 mg) are required, which further lead to non-specific toxicities and also contribute toward cancer resistance. Thus, to overcome these challenges, pazopanib-loaded PEGylated nanoliposomes were developed and evaluated against pancreatic cancer cell lines. The nanoliposomes were prepared by thin-film hydration method, followed by characterization and stability studies. This QbD-enabled process design successfully led to the development of a suitable pazopanib liposomal formulation with desirable properties. The % entrapment of PZP-loaded non-PEGylated and PEGylated nanoliposomes was found to be 75.2% and 84.9%, respectively, whereas their particle size was found to be 129.7 nm and 182.0 nm, respectively. The developed liposomal formulations exhibited a prolonged release and showed desirable physicochemical properties. Furthermore, these liposomal formulations were also assessed for in vitro cell lines, such as cell cytotoxicity assay and cell uptake. These studies confirm the effectiveness of developed liposomal formulations against pancreatic cancer cell lines. The outcomes of this work provide encouraging results and a way forward to thoroughly investigate its potential for PDAC treatment.

In silico study and in vitro antileishmanial and antitrypanosomal evaluation of azopyrazoles and azopyrimidines.

Hydrazones 1-6, azo-pyrazoles 7-9 and azo-pyrimidines 10-15 are compounds that exhibit antibacterial activity. The mode of action and structures of these derivatives have been previously confirmed as antibacterial. In this investigation, biological screening and molecular docking studies were performed for derivatives 1-15, with compounds 2, 7, 8, 14 and 15 yielding the best energy scores (from -20.7986 to -10.5302 kcal/mol). Drug-likeness and in silico ADME prediction for the most potent derivatives, 2, 7, 8, 14 and 15, were predicted (from 84.46 to 96.85%). The latter compounds showed good recorded physicochemical properties and pharmacokinetics. Compound 8 demonstrated the strongest inhibition, which was similar to the positive control (eflornithine) against Trypanosoma brucei brucei (WT), with an EC50 of 25.12 and 22.52µM, respectively. Moreover, compound 14 exhibited the best activity against Leishmania mexicana promastigotes and Leishmania major promastigotes (EC50 =46.85; 40.78µM, respectively).

Comparison of feline and human immunodeficiency virus reverse transcriptase enzymes through chemical screening and computational analysis.

Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.

Design, Synthesis, and Insecticidal Activity of Pyridino[1,2-a]pyrimidines Containing Indole Moeites at the 1-Position.

Research on mesoionic structures in pesticide design has gained significant attention in recent years. However, the 1-position of pyridino[1,2-a]pyrimidine is usually designed with 2-chlorothiazole, 2-chloropyridine, or cyano moieties commonly found in neonicotinoid insecticides. In order to enrich the available pharmacophore library, here, we disclose a series of new pyridino[1,2-a]pyrimidine mesoionics bearing indole-containing substituents at the 1-position. Most of these target compounds are confirmed to have good insecticidal activity against aphids through bioevaluation. In addition, a three-dimensional structure-activity relationship model is established to allow access to optimal compound F45 with an LC50 value of 2.97 mg/L. This value is comparable to the property achieved by the positive control triflumezopyrim (LC50 = 2.94 mg/L). Proteomics and molecular docking analysis suggest that compound F45 has the potential to modulate the functioning of the aphid nervous system through its interaction with neuronal nicotinic acetylcholine receptors. This study expands the existing pharmacophore library for the future development of new mesoionic insecticides based on 1-position modifications of the pyridino[1,2-a]pyrimidine scaffold.

Conventional vs Mechanistic IVIVC: A Comparative Study in Establishing Dissolution Safe Space for Extended Release Formulations.

The use of in vitro-in vivo correlation (IVIVC) for extended release oral dosage forms is an important technique that can avoid potential clinical studies. IVIVC has been a topic of discussion over the past two decades since the inception of USFDA guidance. It has been routinely used for biowaivers, establishment of dissolution safe space and clinically relevant dissolution specifications, for supporting site transfers, scale-up and post approval changes. Although conventional or mathematical IVIVC is routinely used, other approach such as mechanistic IVIVC can be of attractive choice as it integrates all the physiological aspects. In the present study, we have performed comparative evaluation of mechanistic and conventional IVIVC for establishment of dissolution safe space using divalproex sodium and tofacitinib extended release formulations as case examples. Conventional IVIVC was established using Phoenix and mechanistic IVIVC was set up using Gastroplus physiologically based biopharmaceutics model (PBBM). Virtual dissolution profiles with varying release rates were constructed around target dissolution profile using Weibull function. After internal and external validation, the virtual dissolution profiles were integrated into mechanistic and conventional IVIVC and safe space was established by absolute error and T/R ratio's methods. The results suggest that mechanistic IVIVC yielded wider safe space as compared to conventional IVIVC. The results suggest that a mechanistic approach of establishing IVIVC may be a flexible approach as it integrates physiological aspects. These findings suggest that mechanistic IVIVC has wider potential as compared to conventional IVIVC to gain wider dissolution safe space and thus can avoid potential clinical studies.

Unveiling structural and energetic characterization of the emissive RNA alphabet anchored in the methylthieno[3,4-d]pyrimidine heterocycle core.

This study presents a comprehensive theoretical exploration of the fluorescent non-natural emissive nucleobases- mthA, mthG, mthC, and mthU derived from the methylthieno[3,4-d]pyrimidine heterocycle. Our calculations, aligning with experimental findings, reveal that these non-natural bases exert minimal influence on the geometry of classical Watson-Crick base pairs within an RNA duplex, maintaining H-bonding akin to natural bases. In terms of energy, the impact of the modified bases, but for mthG, is also found to be little significant. We delved into an in-depth analysis of the photophysical properties of these non-natural bases. This investigation unveiled a correlation between their absorption/emission peaks and the substantial impact of the modification on the energy levels of the highest unoccupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbital (LUMO). Notably, this alteration in energy levels resulted in a significant reduction of the HOMO-LUMO gap, from approximately 5.4-5.5 eV in the natural bases, to roughly 3.9-4.7 eV in the modified bases. This shift led to a consequential change in absorption and emission spectra towards longer wavelengths, elucidating their bathochromic shift.

Structure-Based Design of Novel Thiazolone[3,2-a]pyrimidine Derivatives as Potent RNase H Inhibitors for HIV Therapy.

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 µM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors.

Design, synthesis and biological evaluation of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives as Mnk1/2 inhibitors.

The Mnk-eIF4E axis plays a crucial role in tumor development, and inhibiting Mnk kinases is a promising approach for cancer therapy. Starting with fragment WS23, a series of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives were designed and synthesized. Among these derivatives, compound 15b showed the highest potency with IC50 values of 0.8 and 1.5 nM against Mnk1 and Mnk2, respectively. Additionally, it demonstrated good selectivity among 30 selected kinases. 15b significantly suppressed MOLM-13 and K562 cell lines growth and caused cell cycle arrest. Furthermore, the Western blot assay revealed that 15b effectively downregulated the downstream proteins p-eIF4E, Mcl-1, and c-myc. Additionally, 15b exhibited remarkable stability in rat plasma and rat and human microsomes. In vivo anti-tumor activity study suggested that treatment with 15b suppressed tumor growth in LL/2 syngeneic models. These findings highlight the potential of 15b as a novel and potent Mnks inhibitor, which deserves further investigation.

Antiviral drug discovery: Pyrimidine entry inhibitors for Zika and dengue viruses.

Vector-borne diseases, constituting over 17 % of infectious diseases, are caused by parasites, viruses, and bacteria, and their prevalence is shaped by environmental and social factors. Dengue virus (DENV) and Zika virus (ZIKV), some of the most prevalent infectious agents of this type of diseases, are transmitted by mosquitoes belonging to the genus Aedes. The highest prevalence is observed in tropical regions, inhabited by around 3 billion people. DENV infects millions of people annually and constitutes an additional sanitary challenge due to the circulation of four serotypes, which has complicated vaccine development. ZIKV causes large outbreaks globally and its infection is known to lead to severe neurological diseases, including microcephaly in newborns. Besides, not only mosquito control programs have proved to be not totally effective, but also, no antiviral drugs have been developed so far. The envelope protein (E) is a major component of DENV and ZIKV virion surface. This protein plays a key role during the virus cell entry, constituting an attractive target for the development of antiviral drugs. Our previous studies have identified two pyrimidine analogs (3e and 3h) as inhibitors; however, their activity was found to be hindered by their low water solubility. In this study, we performed a low-throughput antiviral screening, revealing compound 16a as a potent DENV-2 and ZIKV inhibitor (EC50 = 1.4 µM and 2.4 µM, respectively). This work was aimed at designing molecules with improved selectivity and pharmacokinetic properties, thus advancing the antiviral efficacy of compounds for potential therapeutic use.

A comparative study of two thienopyrimidine Schiff base probes for sequential monitoring of Ga3+ and Pd2.

Two Schiff base probes (S1 and S2) were prepared and synthesized by incorporating thienopyrimidine into salicylaldehyde or 3-ethoxysalicylaldehyde individually, with the aim of detecting Ga3+ and Pd2+ sequentially. Upon chelation with Ga3+, S1 and S2 exhibited fluorescence enhancement in DMSO/H2O buffer. Both S1-Ga3+ and S2-Ga3+ were quenched by Pd2+. The limit of detection for S1 in response to Ga3+ and Pd2+ was 2.86 × 10-7 and 4.4 × 10-9 M, respectively. For S2, the limit of detection for Ga3+ and Pd2+ was 4.15 × 10-8 and 3.0 × 10-9 M, respectively. Furthermore, the complexation ratios of both S1 and S2 with Ga3+ and Pd2+ were determined to be 1:2 through Job's plots, ESI-MS analysis, and theoretical calculations. Two molecular logic gates were constructed, leveraging the response behaviors of S1 and S2. Moreover, the potential utility of S1 and S2 for monitoring Ga3+ and Pd2+ in domestic water was verified.

Molecular docking approach for the design and synthesis of new pyrazolopyrimidine analogs of roscovitine as potential CDK2 inhibitors endowed with pronounced anticancer activity.

Cyclin-dependent kinase 2 (CDK2) is a vital protein for controlling cell cycle progression that is critically associated with various malignancies and its inhibition could offer a convenient therapeutic approach in designing anticancer remedies. Consequently, this study aimed to design and synthesize new CDK2 inhibitors featuring roscovitine as a template model. The purine ring of roscovitine was bioisosterically replaced with the pyrazolo[3,4-d]pyrimidine scaffold, in addition to some modifications in the side chains. A preliminary molecular docking study for the target chemotypes in the CDK2 binding domain revealed their ability to accomplish similar binding patterns and interactions to that of the lead compound roscovitine. Afterwards, synthesis of the new derivatives was accomplished. Then, the initial anticancer screening at a single dose by the NCI revealed that compounds 7a, 9c, 11c, 17a and 17b achieved the highest GI% values reaching up to 150 % indicating their remarkable activity. These derivatives were subsequently selected to undertake five-dose testing, where compounds 7a, 9c, 11c and 17a unveiled the most pronounced activity against almost the full panel with GI50 ranges; 1.41-28.2, 0.116-2.39, 0.578-60.6 and 1.75-42.4 µM, respectively and full panel GI50 (MG-MID); 8.24, 0.6, 2.46 and 6.84 µM, respectively. CDK2 inhibition assay presented compounds 7a and 9c as the most potent inhibitors with IC50 values of 0.262 and 0.281 µM, respectively which are nearly 2.4 folds higher than the reference ligand roscovitine (IC50 = 0.641 µM). Besides, flow cytometric analysis on the most susceptible and safe cell lines depicted that 7a caused cell cycle arrest at G1/S phase in renal cancer cell line (RXF393) while 9c led to cell growth arrest at S phase in breast cancer cell line (T-47D) along with pronounced apoptotic induction in the mentioned cell lines. These findings afforded new anticancer pyrazolo[3,4-d]pyrimidine, roscovitine analogs, acting via CDK2 inhibition.

Novel Pyrido[4,3-d]pyrimidine Derivatives as Potential Sterol 14α-Demethylase Inhibitors: Design, Synthesis, Inhibitory Activity, and Molecular Modeling.

Thirty-four new pyrido[4,3-d]pyrimidine analogs were designed, synthesized, and characterized. The crystal structures for compounds 2c and 4f were measured by means of X-ray diffraction of single crystals. The bioassay results showed that most target compounds exhibited good fungicidal activities against Pyricularia oryzae, Rhizoctonia cerealis, Sclerotinia sclerotiorum, Botrytis cinerea, and Penicillium italicum at 16 µg/mL. Compounds 2l, 2m, 4f, and 4g possessed better fungicidal activities than the commercial fungicide epoxiconazole against B. cinerea. Their half maximal effective concentration (EC50) values were 0.191, 0.487, 0.369, 0.586, and 0.670 µg/mL, respectively. Furthermore, the inhibitory activities of the bioactive compounds were determined against sterol 14α-demethylase (CYP51). The results displayed that they had prominent activities. Compounds 2l, 2m, 4f, and 4g also showed better inhibitory activities than epoxiconazole against CYP51. Their half maximal inhibitory concentration (IC50) values were 0.219, 0.602, 0.422, 0.726, and 0.802 µg/mL, respectively. The results of molecular dynamics (MD) simulations exhibited that compounds 2l and 4f possessed a stronger affinity to CYP51 than epoxiconazole.

Design, synthesis, and bioevaluation of SOS1 PROTACs derived from pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor.

Oncogenic KRAS mutations drive an approximately 25 % of all human cancers. Son of Sevenless 1 (SOS1), a critical guanine nucleotide exchange factor, catalyzes the activation of KRAS. Targeting SOS1 degradation has engaged as a promising therapeutic strategy for KRAS-mutant cancers. Herein, we designed and synthesized a series of novel CRBN-recruiting SOS1 PROTACs using the pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor as the warhead. One representative compound 11o effectively induced the degradation of SOS1 in three different KRAS-mutant cancer cell lines with DC50 values ranging from 1.85 to 7.53 nM. Mechanism studies demonstrated that 11o-induced SOS1 degradation was dependent on CRBN and proteasome. Moreover, 11o inhibited the phosphorylation of ERK and displayed potent anti-proliferative activities against SW620, A549 and DLD-1 cells. Further optimization of 11o may provide us promising SOS1 degraders with favorable drug-like properties for developing new chemotherapies targeting KRAS-driven cancers.

Discovery of BIO-8169─A Highly Potent, Selective, and Brain-Penetrant IRAK4 Inhibitor for the Treatment of Neuroinflammation.

Interleukin receptor associated kinase 4 (IRAK4) plays an important role in innate immune signaling through Toll-like and interleukin-1 receptors and represents an attractive target for the treatment of inflammatory diseases and cancer. We previously reported the development of a potent, selective, and brain-penetrant imidazopyrimidine series of IRAK4 inhibitors. However, lead molecule BIO-7488 (1) suffered from low solubility which led to variable PK, compound accumulation, and poor in vivo tolerability. Herein, we describe the discovery of a series of pyridone analogs with improved solubility which are highly potent, selective and demonstrate desirable PK profiles including good oral bioavailability and excellent brain penetration. BIO-8169 (2) reduced the in vivo production of pro-inflammatory cytokines, was well tolerated in safety studies in rodents and dog at margins well above the predicted efficacious exposure and showed promising results in a mouse model for multiple sclerosis.

RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

Discovery of 9H-pyrimido[4,5-b]indole derivatives as dual RET/TRKA inhibitors.

Aberrant RET kinase signaling is activated in numerous cancers including lung, thyroid, breast, pancreatic, and prostate. Recent approvals of selective RET inhibitors, pralsetinib and selpercatinib, has shifted the focus of RET kinase drug discovery programs towards the development of selective inhibitors. However, selective inhibitors invariably lose efficacy as the selective nature of the inhibitor places Darwinian-like pressure on the tumor to bypass treatment through the selection of novel oncogenic drivers. Further, selective inhibitors are restricted for use in tumors with specific genetic backgrounds that do not encompass diverse patient classes. Here we report the identification of a pyrimido indole RET inhibitor found to also have activity against TRK. This selective dual RET/TRK inhibitor can be utilized in tumors with both RET and TRK genetic backgrounds and can also provide blockade of NTRK-fusions that are selected for from RET inhibitor treatments. Efforts towards developing dual RET/TRK inhibitors can be beneficial in terms of encompassing more diverse patient classes while also achieving blockade against emerging resistance mechanisms.

Design and one-pot synthesis of new substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine as potential antitumor agents: in vitro and in vivo studies.

A new efficient and versatile one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives has been developed. It is based on a multistep cascade reaction from 2-aminothiophenes and 2-hydroxy-4-oxobut-2-enoic acids, and derivatives of cyanoacetic acid catalyzed by diisopropylethylamine. As a result, novel pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives (21 compounds) were synthesized in a mild reaction conditions with a high yield. The structures of the developed compounds were confirmed by NMR and elemental analysis. The influence of electron-withdrawing or electron-donor substituents on the antitumor activity of the developed compounds has been identified. In vitro screening analysis of 21 compounds revealed six lead candidates (12aa, 12dc, 12hc, 12ic, 12lb, and 12mb) that demonstrated the most significant antitumor activity against B16-F10, 4T1 and CT26 cells. Necrosis/apoptosis assay showed that apoptosis was the predominant mechanism of cell death. Molecular docking analysis revealed several potential targets for tested compounds, i.e. phosphatidylinositol 5-phosphate 4-kinase (PI5P4K2C), proto-oncogene serine/threonine-protein kinase (Pim-1), nicotinamide phosphoribosyltransferase (NAMPT) and dihydrofolate reductase (DHFR). The lead compound (12aa) can effectively induce cell apoptosis, possesses a high yield (98 %) and requires low-cost starting chemicals for its synthesis. In vivo experiments with melanoma-bearing mice confirmed that 12aa compound resulted in the significant tumor inhibition on 15 d after the therapy. In particular, tumor volume was ∼0.19 cm3 for 50 mg/kg versus ∼2.39 cm3 in case of untreated mice and tumor weight was ∼71.6 mg for 50 mg/kg versus ∼452.4 mg when considered untreated mice. Thus, our results demonstrated the high potential of the 12aa compound in the treatment of melanoma and can be recommended for further preclinical studies.

Design, synthesis, and biological evaluation of 2,4-diaminopyrimidine derivatives as potent Hematopoietic Progenitor Kinase 1 (HPK1) inhibitors.

HPK1 also referred to as MAP4K1, belongs to the category of mammalian STE20-like protein serine/threonine kinases. Its physiological function involves the down-regulation of T cell signals, and it is regarded as a new immune checkpoint of tumor immunology. In this study, we commenced our investigation with the hit compounds, focusing the efforts on structural optimization and SAR exploration to identify a novel class of 2,4-diaminopyrimidine HPK1 inhibitors. Notably, compound 14g exhibited a remarkable inhibitory effect on HPK1 kinase (IC50 = 0.15 nM), significantly suppressed the phosphorylation of the downstream adaptor protein SLP76 (pSLP76 IC50 = 27.92 nM), and effectively stimulated the secretion of the T cell activation marker IL-2 (EC50 = 46.64 nM). In vitro microsomal stability assay, compound 14g showed moderate stability in HLMs with T1/2 = 38.2 min and CLint = 36.4 µL·min-1·mg-1 proteins. In vivo pharmacokinetic studies, compound 14g demonstrated heightened plasma exposure (AUC0-inf = 644 ng·h·mL-1), extended half-life (T1/2 = 9.98 h), and reduced plasma clearance (CL = 52.3 mL·min-1·kg-1) compared to the reference compound after a single intravenous dose of 2 mg/kg in rats. These results indicated that compound 14g emerged as a promising inhibitor of HPK1.